The wavelength dependence of the incoherent point spread function in a
wide-field microscope was investigated experimentally. Dispersion in
the sample and optics can lead to significant changes in the point spr
ead function as wavelength is varied over the range commonly used in f
luorescence microscopy. For a given sample, optical conditions can gen
erally be optimized to produce a point spread function largely free of
spherical aberration at a given wavelength. Unfortunately, deviations
in wavelength from this value will result in spherically aberrated po
int spread functions. Therefore, when multiple fluorophores are used t
o localize different components in the same sample, the image of the d
istribution of at least one of the fluorophores will be spherically ab
errated. This aberration causes a loss of intensity and resolution, th
ereby complicating the localization and analysis of multiple component
s in a multi-wavelength image. We show that optimal resolution can be
restored to a spherically aberrated image by constrained, iterative de
convolution, as long as the spherical aberration in the point spread f
unction used for deconvolution matches the aberration in the image rea
sonably well. The success of this method is essentially independent of
the initial degree of spherical aberration in the image. Deconvolutio
n of many biological images can be achieved by collecting a small libr
ary of spherically aberrated and unaberrated point spread functions, a
nd then choosing a point spread function appropriate for deconvolving
each image. The co-localization and relative intensities of multiple c
omponents can then be accurately studied in a multi-wavelength image.